Neural crest cells invade the cephalic mesoderm.

<p>(A) Schematic representation of the chick neural fold replacement by its quail counterpart (red) performed on 5/6-somite stage quail and chick embryos. (B–F) Transverse sections of quail–chick chimeras at 16 (B,C,D), 18 (E) and 21 (F) somite-stages, at the level of the future first branchial arch were hybridized with the <i>MyoR</i> probe followed by immunohistochemistry using the QCPN mAb. (C) is a higher magnification of (B). (B,C,D) corresponds to sections from the same embryo, (B,C) being slightly (80 µm) more rostral than (D). The asterisk in B marks the position of the graft. (B–D) At the future first branchial arch level, the QCPN-positive cells progressively invaded the cephalic mesoderm expressing <i>MyoR,</i> in a rostral to caudal manner. At 18 and 21 somite-stages (E,F), QCPN positive cells are observed inside the <i>MyoR</i>-positive domains. a, aortic arch; ecto, ectoderm; endo, pharyngeal endoderm; mes, mesencephalon; ph, pharynx.</p

<i>Scleraxis</i> expression is lost in the absence of differentiated muscles in the first branchial arch of E15.5 <i>Tbx1</i>^−/− mice.

<p>Adjacent saggital sections of either E15.5 wild-type (A,C,E) or E15.5 <i>Tbx1</i>^−/− mutant mice (B,D,F) were hybridized with <i>mMyoD</i> (A–D) or <i>mScleraxis</i> (E,F) probes. (E,F) After in situ hybridization with a mouse <i>Scleraxis</i> probe (blue), differentiated myofibres were detected using MF20 antibody (light brown). (C,D) are higher magnification of A,B), respectively, focusing on the mandible. <i>Tbx1</i>^−/− mutant mice display loss of branchiomeric muscles, highlighted by the absence of both <i>mMyoD</i> expression (B,D) and MF20 labelling (F) in the mandibula, compared to the wild type situation (A,C,E). The anterior digastric muscle is arrowed in the control mandible (C,E), while residual muscle masses are indicated by an arrow (D,F) in similar mandibular regions of <i>Tbx1^−/−</i> mutant mice. Non-branchiomeric muscles are not affected in E15.5 <i>Tbx1</i>^−/− embryos. (E) <i>Scleraxis</i> expression is observed in tendons associated with the anterior digastric muscle in the wild type situation (arrowheads). (F) <i>Scleraxis</i> expression is lost in the absence of muscles in E15.5 <i>Tbx1^−/−</i> mutant mice, while <i>Scleraxis</i> expression is normally associated with non-branchiomeric muscles (green arrowheads). Mb, mandibular, mx, maxillary; t, tongue.</p

Comparison of <i>MyoR</i> and <i>MyoD</i> expression domains during chick first branchial arch development.

<p>(A) Lateral view of a HH20 chick embryo hybridized with a <i>MyoR</i> probe. HH20 (B,C), HH22 (D,E) HH24 (F,G) and HH26 (H,I) embryos were frontally sectioned at the head level. The plane of section is indicated in the panel A. Adjacent sections from each stage were hybridized with DIG-labelled antisense probes for either <i>MyoR</i> (B,D,F,H) or <i>MyoD</i> (C,E,G,I). <i>MyoR</i> delineates the myogenic core of the first branchial arch (B,D,F) and is subsequently expressed in all branchiomeric muscles (H). <i>MyoD</i> transcripts are first detected in a lateral sub-region of the <i>MyoR</i> domain at HH20 (B,C, arrows) and HH22 (D,E, arrows), then spread progressively from lateral to medial regions to overlap with the <i>MyoR</i> domain in branchiomeric muscles (E,G,I). (D–G) Arrows point to the lateral domains of the core, while arrowheads show the medial domain of the core. (H,I) Arrows indicate the branchiomeric muscles expressing both <i>MyoR</i> (H) and <i>MyoD</i> (I). (A) Arrowheads indicate the hypaxial lips at the interlimb level, expressing <i>MyoR</i>. BA1, first branchial arch, di, diencephalon; e, eye; fl, forelimb; hl, hindlimb; mb, mandibular arch; mes, mesenchephalon; MR, medial rectus; mx, maxillary arch; ph, pharynx; tel, telencephalon; VO, ventral oblique; VR, ventral rectus.</p

Comparison of <i>MyoR</i> and <i>MyoD</i> expression in extra-ocular muscles.

<p>HH22 (A,B), HH24 (C,D), HH26 (E,F), HH30 (G,H) chick embryos were frontally sectioned at the head level. Adjacent sections from each stage were hybridized with DIG-labelled antisense probes for either <i>MyoR</i> (A,C,E,G) or <i>MyoD</i> (B,D,F,H). E12.5 mouse embryos were sagitally sectioned at the head level and hybridized with DIG-labelled antisense probes for either <i>mMyoR</i> (I) or <i>mMyoD</i> (J). In the chick embryo, At HH22, the ventral oblique is the first ocular muscle to express <i>MyoR</i> and <i>MyoD</i> (A,B). At HH 24, <i>MyoR</i> transcripts are observed faintly in the dorsal rectus, strongly in lateral rectus (C), while <i>MyoD</i> is expressed in both dorsal rectus and lateral rectus muscles and in branchiomeric muscles (D). At HH26 stage, the ventral and dorsal obliques harbour strong <i>MyoR</i> (E) and <i>MyoD</i> (F) expression. At HH30, when the medial and ventral rectus muscles are individualized, <i>MyoD</i> is expressed in both muscles, while <i>MyoR</i> is expressed in ventral rectus but not in medial rectus (G,H). (H) The innervation is labelled with HNK1 antibody, two arrowheads point to nerves. The optic nerve is also labelled in light brown with the HNK1 antibody. (G) The absence of <i>MyoR</i> expression in the medial rectus is indicated by arrows. (I,J) In E12.5 mouse embryos, extra ocular muscles, labelled by white asterisks (J) expressed both <i>mMyoR</i> (I) and <i>mMyoD</i> (J). di, diencephalon; DO, dorsal oblique; DR, dorsal rectus; LR, lateral rectus; mBA1, first branchial arch muscles; MR, medial rectus; mx, maxillary arch; ON, optic nerve; ph, pharynx; VO, ventral oblique; VR, ventral rectus.</p

<i>Scleraxis</i> expression is normally established in the absence of differentiated muscles in the first branchial arch of E12.5 <i>Tbx1</i>^−/− mice.

<p>Adjacent saggital sections of either wild-type (A,C,E) or <i>Tbx1</i>^−/− mutant mice (B,D,F) were hybridized with <i>mMyoD</i> (A,B) or <i>mScleraxis</i> (C–F) probes. (C–F) After in situ hybridization with a mouse <i>Scleraxis</i> probe, the differentiated myofibres were detected using MF20 antibody. <i>Tbx1</i>^−/− mutant mice display a loss of branchiomeric-derived muscles, highlighted by the absence of <i>mMyoD</i> expression (B, black arrow) and MF20 labelling (D, F black arrows) compared to the wild type situation (A,C,E, black arrows). Despite the absence of branchiomeric muscles, <i>mScleraxis</i> expression pattern remains unchanged in mutant mice (D,F black arrowheads) compared to wild-type (C,E black arrowheads). Green arrows (A,B) and arrowheads (C,D) point to the non-affected extraocular muscles and tendons, respectively in control (A,C) and <i>Tbx1</i>^−/− mutant mice (B,D). (E,F) are high magnifications of (C,D) respectively. The open arrow in F indicates <i>Scleraxis</i> domain that has spread to the space left by the absent muscle.</p

Head tendons express <i>Scleraxis</i> and are of quail origin.

<p>(A) In situ hybridization to HH24 embryos with the <i>Scleraxis</i> probe. Adjacent frontal sections of HH24 (B,C) and HH26 (D–H) embryos were hybridized with the <i>Scleraxis</i> (B,D,F,G) and <i>MyoD</i> (C,E,H) probes were followed by an immunohistochemistry using the MF20 antibody to reveal differentiated muscle fibres. MF20 is visible in (F,G). (A,B) Arrows point to the <i>Scleraxis</i> expression domain in tendon primordia at HH24. At HH26, <i>Scleraxis</i> labels branchiomeric tendons (D,E) and eye tendons (F–H). (I–M) Adjacent saggital sections of HH28 quail-chick chimeras, were hybridized with the <i>MyoD</i> (I,K) and <i>Scleraxis</i> (J,L,M) probes followed by immunohistochemistry using the QCPN antibody. QCPN is visible in (I,JM). The <i>Scleraxis</i>-positive tendons associated with the extra ocular muscle (I,J) or with a jaw operating–muscle (K–M) are of quail origin. (M) is a higher magnification of (L).</p

Neural crest cells visualized with <i>AP2α</i> expression are observed inside the mesodermal core of the first branchial arch.

<p>Adjacent transverse sections of chick embryos at the level of the first branchial arch at 22 (A,B), 29 (C,D) and 32 (E,F) somite-stages were hybridized with <i>MyoR</i> (A,C,E) and <i>AP2</i>α (B,D,F) probes. The mesodermal core is visualized with <i>MyoR</i> expression (A,C,E). <i>AP2α</i> is expressed in all neural crest cells within the arch and in the surface ectoderm (B,D,F). The <i>AP2α</i>-positive surface ectoderm is arrowed in (B,D,F). <i>AP2 α</i> positive cells are also observed inside the <i>MyoR</i>-positive domain (B,D,F). The inset in F shows an enlargement of the mesodermal core, where the <i>AP2 α-</i>positive cells are indicated by arrowheads. Frontal sections of E9.5 mouse embryos from <i>Myf5-nlacZ</i> mice were immunostained using an anti-β-galactosidase antibody to visualise <i>mMyf5</i> expression (G, green) and an anti-AP2 antibody to detect mAP2<i>α</i> location (H, red). I is a merged picture of G and H. AP<i>2α</i>-positive cells (red) are observed in the mesodermal core delineated by <i>Myf5</i>-postive cells (green). Hoechst staining in blue indicate nuclei. a, aortic arch; BA1, first branchial arch; BA2, second branchial arch; ecto, surface ectoderm; endo, pharyngeal endoderm; No, notochord; ph, pharynx.</p

Mechanical signals are required for normal tendon gene response following tendon injury.

<p><b>(A)</b> Botox or physiological saline injections in Gastrocnemius muscles and tendon injury in adult mice. (<b>B</b>) RT-q-PCR analyses of tendons, 2 weeks after tendon injury and Botox injection in muscles. The mRNA levels of tendons following injury and physiological saline injection in muscles were normalized to 1. The errors bars represent the standard error of the means of 10 and 11 biological samples of injured tendons after physiological saline or Botox injections, respectively. The <i>p</i> values were calculated using the Mann-Whitney test. The mRNA levels of the <i>Egr1</i>, <i>Scx</i>, <i>Tnmd</i>, <i>Col1a1</i>, <i>Col1a2</i> and <i>Tgfb2</i> genes were all significantly decreased in reduced movement conditions compared to controls during the healing process, following tendon injury.</p

EGR1 forced expression in tendons prevents the diminution of tendon gene expression during tendon healing in reduced mechanical load.

<p>(<b>A</b>) Description of the experimental design for sonoporation. 10 μg of EGR1 encoding plasmid and 3.75 μl of MicroMarker microbubbles were injected in the Achilles tendon sheath. Tendons were then stimulated by ultrasound at 1 MHz during 10 minutes at 200 kPa, 40% duty cycle and 10 kHz pulse repeating frequency. The day after EGR1 sonoporation, a surgical lesion of the Achilles tendon was performed followed by a Botox or physiological saline solution injection in the muscle. Two weeks after treatment, tendons were harvested for analyses by RT-q-PCR. (<b>B</b>) RT-q-PCR analysis of tendon gene expression in EGR1-sonoporated tendons versus control-tendons, following tendon injury in immobilization conditions. The mRNA levels of control tendons following injury and Botox injection in muscles were normalized to 1. The error bars represent standard errors of the mean of 6 biological samples of injured tendons of Botox-injected legs in the absence of EGR1 and 10 biological samples of injured tendons of Botox-injected legs in presence of ectopic EGR1. The <i>p</i> values were calculated using the Mann-Whitney test. The mRNA levels of <i>Egr1</i>, <i>Scx</i>, <i>Tnmd</i>, <i>Col1a2</i> and <i>Tgfb2</i> were increased in Egr1-sonoporated tendons compared to control tendons, following tendon injury and Botox injection in muscles.</p

Reduced mechanical input induces a diminution of <i>Egr1</i> and <i>Scx</i> expression in adult tendons.

<p>(<b>A</b>) Botox or physiological saline injections in Gastrocnemius muscles of adult mice. (<b>B,C</b>) LacZ staining (reflecting Egr1 expression) in tendons, 1 week following physiological saline or Botox injection in Egr1^Lacz/+ adult mice. (<b>D</b>) RT-q-PCR analyses of tendons, one or two weeks after Botox or physiological saline injections in muscles in adult mice. The mRNA levels of tendons following Botox injection were compared to those of tendons with physiological saline injection. Errors bars represent the standard deviations of 5 (one week) and 7 (two weeks) biological samples. The mRNA levels of <i>Egr1</i>, <i>Scx</i>, <i>Col1a2</i>, <i>Tgfb2</i> genes were decreased one or two weeks after Botox injection compared to physiological saline injection. <i>Tnmd</i> mRNA levels were not significantly decreased after Botox injection. The <i>p</i> values were calculated using the Wilcoxon test.</p